Contact PD/PI: Stanley, Sarah A Project Summary. The bacterial pathogen Mycobacterium tuberculosis is highly resistant to oxidative stress encountered in the host, however defense mechanisms remain poorly characterized. This proposal seeks to characterize a nanocompartment system in M. tuberculosis that we propose contributes to defense against oxidative stress. Nanocompartments are protein-based organelles that encapsulate an enzymatic cargo, often an enzyme related to oxidative defense. Although genes encoding nanocompartments are widespread in bacteria and archaea, their endogenous functions are not well understood and it is not clear what benefit the encapsulation of specific enzymes provides. We have discovered that M. tuberculosis has a bacterial nanocompartment system that is required for defense against oxidative stress. This system consists of the encapsulin protein Cfp29 and the cargo protein DypB, a dye decolorizing peroxidase. Our hypothesis is that the M. tuberculosis DypB nanocompartment system is required for resisting oxidative stresses encountered in host macrophages. Building on preliminary data in which we show that DypB encapsulin mutants are attenuated for growth in macrophages, and that these mutants are also susceptible to H2O2 at pH 4.5 in axenic culture we test this hypothesis in three aims. 1) Determine whether encapsulation promotes DypB stability and function; 2) Determine whether the M. tuberculosis DypB encapsulin system is required for defense against lipid peroxides; 3) Determine the role of the DypB nanocompartment in virulence of M. tuberculosis. If successful, the proposed experiments will provide the first link between a nanocompartment system and bacterial virulence, advancing our understanding of how M. tuberculosis, and possibly other pathogens, defend against diverse oxidative stresses encountered in the host. In addition, these studies will provide insights into the function of encapsulin systems and the specific role of the shell protein. Finally, these studies will advance our understanding of the endogenous functions of DyP peroxidases, which are widespread throughout bacteria, archaea, and eukaryotes Page 6 Project Summary/Abstract